Conditional expression of Sox17 in respiratory epithelial cells of the adult mouse lung induced proliferation and reversibly respecified alveolar type II epithelial cells to express markers characteristic of the differentiated bronchiolar epithelium, supporting the concept that a subset of mature respiratory epithelial cells possesses remarkable phenotypic plasticity and progenitor cell capabilities. Activation of this progenitor-like behavior in respiratory epithelial cells by Sox17 was associated with increased expression of Sca-1 and multiple genes that promote cell cycle reentry/progression. Sox17 decreased expression of cell cycle inhibitors in vivo and interacted with Smad3 to inhibit TGF-β1/Smad3-mediated transcriptional responses in vitro. Together, these data provide insight into the mechanisms by which Sox17 stimulates respiratory epithelial progenitor cell behavior and lineage respecification in the mature lung.
Expression of Sox17 reprogrammed a subset of mature alveolar type II cells to ectopically express markers characteristic of diverse conducting airway cell lineages, including ciliated, non-ciliated secretory cells, and goblet cells that are not normally detected in the alveolar regions of the lung 
, and led to the formation of highly organized bronchiolar-like structures in the peripheral lung. These results indicate that epithelial cells in the adult lung can serve as multipotent progenitors capable of lineage respecification. In support of this concept, conditional expression of SPDEF, an ETS family transcription factor, in respiratory epithelial cells of adult mice converts Clara cells into goblet cells 
. The notion that Sox proteins have important functions in lineage reprogramming is further supported by the contribution of Sox2 toward induced pluripotency in somatic cells 
. While the mechanisms that govern progenitor cell activation and cell fate respecification remain poorly understood, further analysis of Sox17-induced respiratory epithelial progenitors may provide insight into such processes in the mature lung.
Lineage reprogramming of adult cell types involves the expression of key developmental regulatory genes 
. The finding from previous studies that expression of several transcription factors involved in regulating proximal/distal epithelial cell differentiation in the lung, including TTF-1, Foxa1, Foxa2, and β-catenin, is also increased in the Sox17-induced cell clusters suggests that Sox17 can reinitiate a program typical of the developing lung 
. During endoderm formation in Xenopus, Sox17 regulates expression of Foxa1
, and acts upstream of GATA6 
. Foxa1, Foxa2, and GATA6 influence the expression of differentiated respiratory epithelial cell markers, including CCSP and Foxj1 
. Taken together, these data support the concept that Sox17, which is necessary for endoderm formation, functions upstream of a hierarchy of transcription factors that cooperate in specification of endodermal cells from which respiratory epithelial lineages are derived. Constitutive expression of Sox17 in human embryonic stem cells is sufficient to commit cells to the definitive endoderm lineage, and increased expression of Sox17 is associated with the differentiation of embryonic stem cells toward cells with characteristics of the respiratory epithelium 
. Together, these findings further support an early role for Sox17 in the establishment of endodermal cells that later serve as precursors of lung epithelial lineages.
The induction of proliferation in a subset of bronchiolar cells and alveolar type II cells by Sox17 was associated with increased expression of several cyclin genes known to stimulate cell cycle progression. Among the various cyclin genes induced by Sox17, only cyclin D1 functions in G1 prior to the restriction point of the cell cycle 
. Notably, forced expression of cyclin D1 along with cdk4 is sufficient to reinitiate cell cycle progression in multiple post-mitotic cell types 
. Therefore, the induction of cyclin D1 by Sox17 is likely to play an important role in stimulating mature respiratory epithelial cells to reenter the cell cycle. Our in vitro
studies demonstrated that Sox17 directly activated the human cyclin D1
promoter through a conserved site located at −74 bp relative to the first exon. Given the homology between their HMG domains, Sox and TCF/LEF proteins have similar preferences for consensus binding sequences, and previous studies have demonstrated that cyclin D1
is directly regulated by β-catenin/TCF/LEF through this same site 
. In breast cancer cells, Sox2 interacts with β-catenin to cooperatively regulate the cyclin D1
promoter through the −74 bp binding site as well 
. In addition, Sox17 and Sox4 respectively inhibit and enhance proliferation of colon carcinoma cells through physical interactions with β-catenin and TCF/LEF to modulate protein stability and transcriptional activity, and Sox6 interacts with β-catenin and HDAC1 to repress cyclin D1
promoter activity and proliferation in insulinoma cells 
. Taken together, these findings suggest that Sox proteins and β-catenin/TCF/LEF complexes may compete for common DNA binding sites and that their effects on transcription and cell behavior may depend on relative expression levels, protein interactions, and/or cell type. Although it is unclear whether Sox17 is a transcriptional activator of cyclin D1 in vivo
, our data support a potential mechanism by which Sox17 directly induces cyclin D1
to promote proliferation of a subset of mature respiratory epithelial cells.
Recent studies have shown that post-mitotic cells can reenter the cell cycle by blocking the expression of cyclin-dependent kinase inhibitors, supporting a role for this protein family in maintaining the balance between quiescence and proliferation 
. In the present study, Sox17 decreased the expression of p15
, and p57
in adult mouse lungs, and inhibited TGF-β1/Smad3 transcriptional activity in vitro
. In addition, Sox17 physically interacted with Smad3 and abrogated Smad3 binding to the p15
promoter. These findings support a model in which Sox17 antagonizes TGF-β/Smad-dependent expression of cell cycle inhibitors in mature respiratory epithelial cells, facilitating G1 progression and cell reentry. In addition, Sox17 counteracted repression of the cyclin D1
promoter by TGF-β1 and Smad3. While we favor the notion that Sox17 directly activates the cyclin D1
promoter, it is possible that inhibiting TGF-β/Smad3-mediated repression of cyclin D1
also contributes to its increased expression in CCSPrtTA/tetO-Sox17
mouse lungs. By decreasing expression of inhibitors and increasing expression of positive regulators of the G1 phase of the cell cycle, Sox17 establishes conditions that favor reactivation of proliferation in mature, normally quiescent respiratory epithelial cells.
In the present study, Sox17 interacted with Smad3 and repressed TGF-β1/Smad3 transcriptional activity. Although both the MH1 and MH2 domains of Smad3 bound Sox17, the strongest interaction was localized to the linker region and MH2 domains. The linker region of Smad3 contains a transactivation domain and the MH2 domain modulates transcription, whereas the MH1 domain of Smad3 regulates DNA binding and transcription. Thus, Sox17 interaction with these domains is consistent with the antagonistic effects of Sox17 on Smad3 DNA binding and transcriptional activity. While that amino acids 129–359 of Sox17 appear to mediate binding to Smad3, the N-terminus of Sox17 is necessary to antagonize Smad3 activity. Inhibition of β-catenin/TCF transcriptional activity is also dependent on the N-terminus of Sox17, wherein the HMG domain is required for interaction with TCF factors to influence their protein stability 
. Together, these findings indicate that the N-terminus of Sox17 containing the HMG domain is important for influencing the transcriptional responses of signaling pathways and that there may be multiple regions of Sox17 that contribute to complex protein interactions.
Sox17 initiated progenitor cell behavior in respiratory epithelial cells when ectopically expressed at high levels in the adult lung. While these studies provide insight into the effects of Sox17 on reprogramming mature respiratory epithelial cells, it is unclear whether they reflect a physiological role during development or repair. Since Sox17 is highly expressed in the endoderm, which gives rise to the respiratory epithelium, Sox17 likely plays an early role in specification of endodermal precursor cells prior to emergence of the lung primordium. In the lung, Sox17 is expressed in mesenchymal cells during branching morphogenesis and in pulmonary endothelial cells later in development and in the adult 
. However, Sox17 mRNA was not detected in respiratory epithelial cells and immunohistochemical staining for endogenous Sox17 was not observed in the lung epithelium in the present study 
. A number of Sox family members are expressed at high levels in various cell types in the developing and mature lung, including Sox2, 4, 7, 9, 11, 17, and 18 
. However, whether Sox proteins have distinct or redundant functions during lung formation and repair remains to be elucidated. Our data shows that the ability of Sox17 to induce progenitor cell behavior is mediated, at least in part, by activating cyclin D1 and decreasing TGF-β-responsive cell cycle inhibitor expression to promote proliferation. As the transcriptional pathways that regulate cell proliferation and differentiation during lung formation are reactivated during regeneration of pulmonary cell lineages following injury, determining how Sox proteins and other transcription factors integrate signals from multiple pathways is important toward understanding the regulatory mechanisms that control lung development and homeostasis.